High-purity inhalable particles of insulin and insulin analogues, and high-efficiency methods of manufacturing the same

ABSTRACT

A high-purity inhalable insulin material, used for preparing a pulmonary pharmaceutical product, includes insulin particles having a particle size at the micrometer level and having the following characteristics: (i) the purity of insulin is not less than 96% on the dried basis; (ii) the total amount of insulin-related impurities is not more than 2%; (iii) the total amount of solvent impurities, which is not a co-solvent formulation component for a pulmonary product, is not more than 0.03%; and (iv) the total amount of non-solvent impurities is not more than 0.3%. Up to 99% by volume of the insulin particles in the inhalable insulin have a particle size of less than 5 μm, based on the total volume of the insulin particles. A high-efficiency method prepares high-purity inhalable insulin material. The yield rate for the high-efficiency method is 75 to 85% or more.

FIELD

Embodiments of the invention relate in general to pulmonary delivery ofhigh-purity human insulin and/or a human insulin analogue, and ahigh-efficiency process for manufacturing particles of insulin (e.g.,human insulin and/or a human insulin analogue) for pulmonary delivery.Aspects of embodiments of the disclosure also relate in general tocompositions including insulin particles (e.g., human insulin particlesand/or human insulin analogue particles) having improved particlecharacteristics.

BACKGROUND

Growing attention has been given to the potential of a pulmonarydelivery route for non-invasive administration and systemic delivery oftherapeutic agents (mainly peptides and proteins) because the lungs arecapable of providing a large absorptive surface area (up to 100 m²) andhave absorptive mucosal membranes that are very or extremely thin (e.g.,have a thickness of about 0.1 μm-0.2 μm) and have good blood supply. Avery thin alveolar-capillary and a bronchial-capillary barrier on asurface of the lungs allows for rapid uptake of human insulin particlesinto a subject's bloodstream, at a rate similar to that achieved withthe rapid-acting human insulin analogue, which is an altered form ofhuman insulin that is different from human insulin that occurs innature, but still functions in the human body in a manner similar tohuman insulin, but with better performance in terms of glycemic control.

Insulin formulations may be administered by subcutaneous or intravenousinjection. Inhaled insulin appears to be as effective as injectedshort-acting insulin. Pulmonary delivery technology was developed sothat inhaled insulin can effectively reach the lung capillaries where itis absorbed.

Human lung airways contain bronchial tubes, which are impermeable toinsulin, as well as alveoli. Inhaled insulin can be absorbed through thealveoli and enter into the circulation system. Inhaled asthmamedications deposit before reaching the alveoli. Devices can deliverhuman insulin particles via slow and even breaths into the alveoli, andthe human insulin can be released into the circulation system.

Inhaled human insulin may be used for pre-meal insulin delivery inpeople with type I and/or II diabetes. Its use may also facilitate theearly introduction of insulin therapy to people who are averse toinsulin injections due to reactions, such as inflammation, bruising,anxiety, and the like.

SUMMARY

According to an embodiment of the present disclosure, a method ofpreparing an inhalable insulin (e.g., human insulin, animal insulin,and/or a human insulin analogue) suitable for pulmonary deliveryincludes: dissolving an insulin raw material in an acidic solution(e.g., a mixture of water and methanol) to form a dissolved insulinsolution; titrating the dissolved insulin solution with a buffersolution to form a suspension comprising insulin particles; stabilizingthe insulin particles with ethanol; concentrating; and washing withethanol and concentrating to obtain insulin particles with a particlesize at the micrometer level suitable for pulmonary pharmaceuticaldrugs.

The acidic solution may include water, an organic solvent, e.g.methanol, or a mixture thereof.

The acidic solution may include the organic solvent in an amount of 10to 90 vol %, based on the total volume of the acidic solution.

The acidic solution may include the organic solvent in an amount ofgreater than 0 to 90 vol % of the total volume of the acidic solution.

The organic solvent may include an alcohol.

The alcohol may include methanol, ethanol, or a mixture thereof.

The buffer solution may have a pH of 3 to 10.

The stabilizing of the micronized insulin particles may include adding astabilizing agent to the suspension.

The stabilizing agent may have a neutral pH and may be miscible withwater.

The stabilizing agent may include an alcohol, a ketone, or a mixturethereof.

The stabilizing may increase the yield of the micronized insulinparticles.

The micronized insulin particles may be prepared at a pH of 3 to 9.

The micronized insulin particles may be prepared at a pH of 4.5 to 7.5.

The micronized insulin particles may include substantially sphericalparticles having a volume mean diameter of about 1 to 2 μm (e.g., 1.2 to2 μm).

The micronized insulin particles may include up to 99 vol % of particleshaving a particle size of less than 5 μm, based on the total volume ofthe micronized insulin particles.

The acidic solution may have a pH range of 1.0 to 3.0. For example, theacidic solution may have a pH in a range of 1.5 to 2.5 (e.g., 1.5 to2.5).

The acidic solution may have a pH of about 2 and may include water and10 vol % to 90 vol % of an organic solvent including methanol, ethanol,or a mixture thereof, based on the total volume of the acidic solution.

The micronized insulin particles may be substantially spherical in shapeand may have a particle size of less than 5 μm.

The micronized insulin particles may include an insulin including humaninsulin, an animal insulin, an insulin analogue, or a mixture thereof.

The insulin analogue may include insulin aspart, insulin glargine, or amixture thereof.

The dissolving, the titrating, and/or the stabilizing procedures may beperformed at room temperature.

The insulin raw material may include a crystalline insulin includingcrystalline human insulin, a crystalline animal insulin, a crystallineinsulin analogue, or a mixture thereof.

The crystalline insulin analogue may include crystalline insulin aspart,crystalline insulin glargine, or a mixture thereof.

According to an embodiment of the present disclosure, micronized insulinparticles include substantially spherical particles comprising aninsulin selected from the group consisting of human insulin, an animalinsulin, an insulin analogue, and a mixture thereof.

The obtained inhalable insulin particles have high purity of insulin(e.g., >98% on the dried basis, e.g., >98% by weight based on the totalweight of the insulin particles on a dried basis) and low impurities,such as, for example: insulin-related impurities are less than 2% on thedried basis, e.g., less than 2% by weight based on the total weight ofthe insulin particles on a dried basis; total amount of solventimpurities are less than 0.03% on the dried basis, e.g., less than 0.03%by weight based on the total weight of the insulin particles on a driedbasis, (where the solvent impurities do not include a co-solventcomponent for the pulmonary drug formulation), and non-solventimpurities are less than 0.3% on the dried basis, e.g., 0.03% by weightbased on the total weight of the insulin particles on a dried basis.

The disclosed method has a high-efficiency. The yield generated for theinsulin particles is in the range of 75-85% by weight, or even higher,based on the total weight of the final product. For example, the yieldof insulin particles may be 75% or more by weight (e.g., 85% or more byweight) based on the total weight of the final product.

The substantially spherical particles may have a volume mean diameter ofabout 1.2 to 2 μm.

Up to 99 vol % of the substantially spherical particles may have aparticle size of less than 5 μm, based on the total volume of themicronized insulin particles.

The insulin analogue may include insulin aspart, insulin glargine, or amixture thereof.

The foregoing description of embodiments of the present disclosure isnot meant to be an exhaustive summary, inasmuch as additional pertinentaspects of the present disclosure will be readily apparent to thoseskilled in the art from the following detailed description, takenindependently or in conjunction with the accompanying drawings andtables, in which one or more embodiments of the invention are describedand shown.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, together with the specification, illustrateembodiments of the present disclosure, and, together with thedescription, serve to explain the principles of the present disclosure.

FIG. 1 is a flow chart illustrating an embodiment of a process formicronizing insulin and/or an insulin analogue.

FIG. 2 is a Scanning Electron Microscopy (SEM) Image of micronized humaninsulin particles prepared according to an embodiment of the presentdisclosure.

FIG. 3 is a graph illustrating a particle size distribution ofmicronized human insulin particles prepared according to the embodimentof FIG. 2.

FIG. 4 is a chart showing an impurity profile of human insulin beforeand after micronizing according to an embodiment of the presentdisclosure.

FIG. 5 is a high-performance liquid chromatography (HPLC) chromatographof dissolved, micronized insulin particles prepared according to anembodiment of the present disclosure.

FIGS. 6 and 7 are charts showing data from an Andersen Cascade Impactorstudy of human insulin particles delivered from a filled canister asprepared according to an embodiment of the present disclosure.

FIG. 8 is a Scanning Electron Microscopy (SEM) image of micronizedinsulin glargine particles prepared according to an embodiment of thepresent disclosure.

FIG. 9 is an HPLC chromatograph of dissolved, micronized insulinglargine particles prepared according to the embodiment of FIG. 8.

FIGS. 10 and 11 are charts showing the results of an Andersen CascadeImpactor study of insulin glargine particles delivered from a filledcanister as prepared according to the embodiment of the presentdisclosure.

FIG. 12 is a Scanning Electron Microscopy (SEM) image of micronizedinsulin aspart particles prepared according to an embodiment of thepresent disclosure.

FIG. 13 is an HPLC chromatograph of dissolved micronized insulin aspartparticles prepared according to the embodiment of FIG. 12.

FIGS. 14 and 15 are charts showing results of an Andersen CascadeImpactor study of insulin aspart particles delivered from a filledcanister as prepared according to the embodiment of the presentdisclosure.

FIG. 16 is an Atom Force Microscopy (AFM) image of human insulinparticles prepared according to a jet milling method.

FIG. 17 is an Atom Force Microscopy (AFM) image of micronized insulinparticles that were prepared as described with respect to Example 2.

DETAILED DESCRIPTION

The following detailed description is provided only for purposes ofillustration of certain specific embodiments of the present disclosureand not for purposes of limiting the scope of the present invention.Alternate embodiments will be readily apparent to those skilled in theart and are intended to be included within the scope of the presentinvention. Also, in the context of the present application, the term“insulin” is used in a broad sense and encompasses any form of insulinor insulin analogue that can be used to treat a human or animal. Forexample, as used herein, the term “insulin” encompasses natural orsynthetic human insulin, natural or synthetic animal insulin, andinsulin analogues (e.g., insulin aspart, insulin glargine, and thelike).

An embodiment of a micronization process for preparing inhalable insulinparticles for pulmonary delivery includes: dissolving an insulin rawmaterial (e.g., a crystalline insulin and/or a crystalline insulinanalogue) in an acidic environment (e.g., dissolving in an acidicsolution to facilitate dissolution of the insulin raw material) to forma dissolved insulin solution; titrating the dissolved insulin solutionwith a buffer solution to form a suspension including insulin particleshaving a particle size at the micrometer level; and adding a stabilizingagent (e.g., an organic solvent and/or a co-solvent) to stabilize theinsulin particles (e.g., to increase the yield of the insulin particlesbefore purification and drying) at 0 to 25° C.; concentrating thesuspension; washing the suspension with a solvent (e.g., ethanol) at 0to 25° C. then concentrating, where washing/concentrating may beconducted multiple times.

Embodiments of the process are conducted at room temperature or lowertemperature and avoid or reduce the introduction of high temperatures(which will cause a reduction of the purity of insulin and also willresult in an increase of impurities), avoid special chemical reagents(which will introduce more non-solvent impurities), avoid long durationprocesses (which will generate more insulin-related impurities even atroom temperature), and avoid mechanical forces such as those introducedby jet milling processes. Some embodiments of the process are performedwithout addition of a polymer (e.g., an excipient polymer, such that asignificant amount of polymer would exist in the obtained insulinparticles) to the acidic environment, including the dissolved insulinsolution and/or the suspension.

Embodiments of the present invention provide a process for theproduction of high-purity inhalable insulin that is suitable forpulmonary delivery. Embodiments of the process utilize raw crystallineinsulin, which may have particle sizes in a sub-millimeter range, toprovide inhalable insulin particles having a particle size in amicrometer range as an active pharmaceutical ingredient (API) forpulmonary delivery having improved characteristics, including morespherical shape, as well as improved smoothness. As described herein,the particle size or particle diameter (e.g., volume mean diameter) maybe measured by a laser diffraction method, unless otherwise specified.

Pulmonary delivery of a drug particle is affected by the characteristicsof the drug particle including particle size, particle shape, surfaceroughness, solubility, flowability, and/or the like. Since inhalableinsulin and/or insulin analogues are an active drug ingredient and notjust a passive carrier, embodiments of the present disclosure maintainor substantially maintain biological activities while micronizing theinsulin and insulin analogues as high-purity insulin particles.

As reported above, the obtained insulin particles will be used toprepare pulmonary pharmaceutical drugs. Besides the particle size, thehigh-purity of insulin and low impurities will be important.

A particle having a particle size (or an aerodynamic diameter) of <5 μmallows for the inhaled drug to be absorbed by the lungs. Particleshaving a suitable aerodynamic diameter or particle size have good flowproperties and are more easily dispersed into the lower airways(bronchial and alveolar regions) in which the absorption into thebloodstream is improved or optimized via alveolar-capillary surfaces ofthe lungs. On the other hand, over-sized drug particles (e.g., particleshaving an aerodynamic diameter or particle size >5 μm) would be mostlycaptured in the upper airways such as the throat and trachea by inertialimpaction. The over-sized particles are substantially not absorbed asthey accumulate in the upper airways, which do not have the thinpenetrable capillaries of the alveoli. The accumulated drug particlesmay trigger the pulmonary defense system, which may prompt macrophagesincrement. The stimulation or excessive stimulation of macrophages maylead to recruitment of other inflammatory cells and may eventuallyproduce secondary tissue damage, regeneration and fibrosis.

Drug particle size may play a determinant role in pulmonary delivery. Tofabricate particles having a particle diameter <5 μm, a number ofsingle-step micronization methods may be used, such as spray drying andmechanical milling technologies, such that after the process, thestarting raw insulin powder particles, which in general have a diameterof millimeter range, have a diameter in a micrometer range for pulmonarydelivery.

However, those processes for micronizing insulin particle involveintroduction of heat at high temperature, lengthy processes, and/orexcipient polymers and special chemical reagents during the insulinmicronization process, which may cause aggregation and loss of activityof the insulin, reduce its purity, or introduce more impurities, andwhich may hinder pharmaceutical manufacturing. In addition, although theexcipient polymer and other special reagents help to stabilize theformulation and increase the solubility during processing, the excipientpolymer and other special reagents may introduce impurities that aredifficult to remove.

It has also been found that micro-particles of insulin are formed bydissolving crystalline insulin at a pH near the isoelectric point of theinsulin, when a polymer is used in the process of forming the insulinmicro-particles. Various suitable types of polymers such as polyethyleneglycol (PEG), polyvinylpyrrolidone (PVP), poly-lactic acid-co-glycolideacid (PLGA), as well as bioadhesive mechanisms, may be used in theprocess. When the polymer is added to the buffer solution, it may helpto further increase the solubility of the crystalline insulin. However,the added polymer may not be efficiently and completely removed afterthe process. The residual polymer that is not removed may reduce drugefficacy, increase toxicity, and increase the level of impurities.

Other processes related to the production of microspheres that containinsulin introduce excipient polymers such as PVP, or PEG to helpdissolve insulin in an acidic environment. Microspheres produced by suchprocesses are exposed to relatively high temperatures that may behazardous or damaging to insulin. At the end of such processes, anorganic solvent (which has low solubility for insulin) for washing thepolymer away may cause agglomeration of small insulin particles. Also,the foregoing organic solvents can denature insulin molecules containedin the microspheres and may also be toxic when administered to humans oranimals.

Some insulin particles obtained by microencapsulation (e.g., by way of asurfactant) of uniform microcrystals of insulin using biodegradablepolymeric materials. Such compositions, however, may have a low insulincontent, for example, an average insulin particle may contain only 10 to30% insulin w/w, based on the total weight of the insulin particle.

Aspects of embodiments of the present disclosure are directed towardovercoming the above-mentioned difficulties and generate high-purityinsulin particles for pulmonary pharmaceutical products An embodiment ofa method of manufacturing an inhalable insulin or insulin analogue mayinclude the following four (4) actions:

(1) Dissolving an insulin raw material (e.g., crystalline insulin orinsulin analogue) in an acidic environment to facilitate dissolution ofthe insulin raw material, thereby forming a dissolved true insulinsolution. The acidic environment may include an acidic solution. Forexample, the acidic environment may include an acidic solution includingwater, an organic solvent (e.g., an alcohol, such as methanol), or amixture thereof.

The behavior of insulin in an acidic environment may be utilized todissolve insulin. In some embodiments, the acidic environment has a pHof about 1.0 to 3.0, for example, 1.5 to 2.5 (e.g., 1.8 to 2.5), toprovide good dissolution conditions.

(2) Titrating the dissolved insulin solution with a buffer solutionuntil the status of a suspension is reached (e.g., until a suspension isobtained). The titrating of the dissolved insulin solution causes thedissolved insulin to precipitate as insulin particles having a suitableparticle size and shape and to form a suspension. As insulinprecipitates the dissolved insulin solution changes from a clear orsubstantially clear solution to a milky and whitish suspension (e.g.,the suspension including micronized insulin particles).

(3) Stabilizing the micronized insulin particles by adding a stabilizingagent (e.g., an organic solvent ethanol) to increase the yield of theinsulin particles. The stabilizing agent may be added at roomtemperature or lower temperature in the range of 0-25° C. Thestabilizing agent (e.g., the organic solvent and, optionally, theco-solvent) utilized may be varied according to the type of insulin andwill be further described in the following section. Then, the suspensionwill be concentrated.

(4) the concentrated suspension of the insulin particles then will bewashed with the same solvent (e.g., the same kind of solvent) at roomtemperature or lower temperature in the range of 0-25° C. Thiswashing/concentrating may be conducted multiple times.

The above process will generate high-purity insulin particles. Theobtained inhalable insulin particles have high purity of insulin(e.g., >98% on the dried basis, e.g., >98% by weight based on the totalweight of the insulin particles on a dried basis) and low impurities,such as, for example: insulin-related impurity is less than 2% on thedried basis, e.g., less than 2% by weight based on the total weight ofthe insulin particles on a dried basis; the amount of total solventimpurities are less than 0.03% on the dried basis, e.g., less than 0.03%by weight based on the total weight of the dried insulin particles(where the solvent is not a co-solvent component for the pulmonary drugformulation), and the amount of non-solvent impurity is less than 0.3%on the dried basis, e.g., less than 0.3% by weight based on the totalweight of the dried insulin particles on a dried basis. As used herein,unless otherwise indicated, the term “dried basis” indicates that theingredient (or component, e.g., insulin particles or insulin) referencedneed not be dried prior to use if due allowance is used with water orother volatile substances present in the quantity taken.

The disclosed method has a high-efficiency, the yield to generate theinsulin particles having particle sizes at the micrometer level is inthe range of 75-85% or even higher (e.g., 75% or more, or 85% or morebased on the total weight of the final product).

Because of the high purity of insulin obtained, and low levels of otherimpurities, the obtained insulin particles can be used for furtherprocessing of pulmonary pharmaceutical products, such as (i) directlyadding, or drying then adding, propellant (HFA 134a or 227) and otherformulation components for a metered dose inhalation (MDI) product, or(ii) drying then mixing with a carrier (such as lactose) for a drypowder inhalation product.

Aspects of embodiments of the present disclosure provide the followingfeatures: high-purity inhalable insulin particles can be manufactured athigh efficiency as compared to other processes.

Embodiments of the novel process for the inhalable particles of insulinand insulin analogues at room temperature for pulmonary deliveryaccording to the present disclosure include the following four majorsteps. First, dissolution of an insulin raw material having a particlesize in the sub-millimeter range in an acidic solvent as a truesolution; second, generating insulin particles with a suitable particlesize by titrating at suitable condition (pH, concentration, mixing, timeetc.); third, stabilizing the generated insulin particles with a solvent(e.g., ethanol), then concentrating at 0-25° C.; and fourth, and,optionally, last, washing with solvent at 0-25° C. and concentrating.

In the first act, the insulin raw material may be dissolved in an acidicenvironment (e.g., an acidic solution) including water and an organicsolvent that is polar, has a small molecular weight and is miscible withwater. Methanol and/or ethanol may be used in the solution in an amountof up to 90 volume percent (vol %), based on the total volume of thesolution, to control the starting solubility of insulin. For example,methanol and/or ethanol may be included in the acidic solution in anamount preferably of approximately 90 vol % (based on the total volumeof the acidic solution), but any amount greater than 0 to up to 90 vol %is contemplated and may be used.

The acidic solution may be placed on top of a stirring plate. Steady,continuous, or substantially continuous stirring, such as 40 to 200rotations per minute (rpm), may be utilized throughout until thesolution becomes completely or substantially completely clear.

In the second act, the stirring speed may be slowed down, such as 30 to100 rpm. The dissolved insulin solution is titrated or slowly titratedwith a buffer solution, such as sodium acetate/acetic acid andprecipitation of the insulin gradually appears as the dissolved insulinsolution changes from a clear or substantially clear solution to a milkywhitish suspension and gradually growing up to suitable particle size.

The particles of insulin and/or insulin analogue may be generated at apH range of 3 to 9, for example, a pH range of 4.5 to 7.5. The buffersolution may be prepared to have same pH range.

In the third act, a stabilizing agent having a neutral pH and attemperature of 0-25° C. and that is miscible with water is utilized.Examples of the stabilizing agent include an alcohol and/or a ketone.For example, the alcohol may include ethanol, or a mixture thereof. Thestabilizing agent stabilizes the micronized insulin particles. Thestabilized suspension then will be concentrated.

In the fourth act, the concentrated suspension will be washed withsolvent such as ethanol at 0-25° C. and further concentrated. Thewashing/concentrating may be repeated multiple times.

The obtained insulin particles can be used for further processing ofpulmonary pharmaceutical products, such as (i) directly adding, ordrying then adding, propellant (HFA 134a or 227) and other formulationcomponents for a metered dose inhalation (MDI) product or (ii) dryingthen mixing with carrier (such as lactose) for a dry powder inhalationproduct.

FIG. 1 is a process flow chart illustrating an embodiment of a methodfor micronizing insulin and/or insulin analogues at room temperature. InFIG. 1, an embodiment of a process 100 for micronizing insulin includesdissolving insulin raw material 102, precipitating (e.g., by titrating)insulin to form and stabilize a suspension 104, separating/stabilizinginsulin 106, and washing and concentrating insulin 108.

Embodiments of the present disclosure will now be described withreference to examples for purposes of illustration. The presentdisclosure, however, is not limited to the examples described herein.

Example 1. Preparation of Inhalable Insulin Particles in a 90 Vol %Methanol Solution

70 mg of biosynthetic human insulin (i. e. from Amphastar FrancePharmaceuticals, S.A.S.) raw material powder was dissolved in 7.7 ml ofan acidic solution having a pH of about 1.9, which is a mixture of 90vol % of methanol 10 vol % water, in a 40 ml vial. The vial was placedon top of a stirring plate and the resultant solution was steadilystirred until the solution was completely dissolved or substantiallyclear to form insulin true solution. Then, the stirring was slowed to aslower mode (e.g., a spinning speed of about 75 rpm), and 1.75 ml of a0.1 M sodium acetate/Acetic Acid (NaAc/HAc) buffer solution having a pHof 5.64 (1.825 g NaAc and 0.165 g Acetic Acid dissolved into 250 mlsolution) was added dropwise to slowly titrate the dissolved insulinsolution. The clear dissolved insulin solution turned into a milky andyellowish suspension including micronized insulin particles. About 10 mlof ethanol (no PH adjusted ethanol) was added to the suspension afterthe titration was completed or substantially completed. The stirring wascontinued for another 30 minutes for stabilizing the particles andhigher yield. The micronized insulin particles were separated from asupernatant of the suspension as a solid by centrifuge and the obtainedsolid was washed with ethanol twice to remove methanol and salt. Thesolid was vacuum dried at room temperature.

FIG. 2 is a scanning electron microscopy (SEM) image showing theinhalable human insulin API produced via the method described withrespect to Example 1. In the present application, all of the SEM imageswere obtained using a JEOL CarryScope JCM-5700 SEM instrument. FIG. 3 isa graph illustrating the particle size distribution of the inhalableinsulin API (micronized insulin) prepared as described with respect toExample 1. It was concluded from FIGS. 2 and 3 that the particle sizesof the inhalable insulin API (micronized insulin) prepared as describedwith respect to Example 1 are suitable for pulmonary delivery, e.g.,have a particle size <5 μm. For example, as can be seen in FIG. 3, theaverage particle size D50 of the micronized insulin of Example 1 wasless than 2 μm. D50 is the maximum particle diameter below which 50 vol% of the sample, based on the total volume of the sample, has a smallerparticle diameter and above which 50 vol % of the sample has a largerparticle diameter.

Example 2. Batch Process for Preparation of Inhalable Insulin Particlesin a 90 Vol % Methanol Solution

1 gram of biosynthetic human insulin API powder (i.e., recombinantinsulin from Amphastar France Pharmaceuticals S.A.S.) was dissolved in110 ml of an acidic solution having a pH of about 1.9 in a mixture of 90vol % of methanol and 10 vol % water, in a 400 ml beaker with acentrifugal stirrer or stirring bar. The resultant solution was stirreduntil the insulin solution was completely dissolved or substantiallycompletely clear to form insulin true solution. Then, the stirring wasslowed to a slower mode (e.g., a spinning speed of about 50 rpm), and 25ml of a 0.1 M sodium acetate/Acetic Acid (NaAc/HAc) buffer solution(having a pH of 5.64) was added dropwise to titrate the dissolvedinsulin solution. The clear dissolved insulin solution turned into amilky and yellowish suspension including micronized insulin particles.After the titration was completed, about 135 ml of ethanol (Neutralethanol) was added to the suspension, and the stirring was continued foranother 30 minutes for stabilizing the particles and higher yield.

The micronized insulin particles were concentrated from the supernatantof the suspension by ultrafiltration with 500 kD modifiedpolyethersulfone membrane from Spectrum Lab at Rancho Dominguez, Calif.First the mixture was concentrated 5-10 ml, which was washed with 100 mlethanol three times to remove methanol and salt. The final volume of thesuspension is 5 to 10 ml. The concentrated suspension was vacuum driedat room temperature for 2.5 hr. The product weight was used to calculatethe recovery rate. The particle size was analyzed using a laserdiffraction particle size analyzer (i.e., the JEOL CarryScope JCM-5700SEM instrument).

The above procedures were repeated four (4) times for batches ofmicronized insulin.

Table 1 shows reproducibility of the recovery rate for the four (4)batches produced as described with respect to Example 2. As can be seenfrom Table 1, the recovery rate for Example 2 is over 86%.

TABLE 1 Process Reproducibility Batch No. Recovery Rate % Batch 1 86.4Batch 2 86.1 Batch 3 86.8 Batch 4 86.2 Average 86.4 Standard Deviation0.3 Relative Standard Deviation, % 0.4%

Table 2 shows the reproducibility of the particle size distribution ofthe micronized human insulin particles produced in the batches ofExample 2. It was concluded from Table 2 that the particles sizes of themicronized insulin prepared as described with respect to Example 2 aresuitable for pulmonary delivery, e.g., having a particle size <5 μm. Forexample, as can be seen in Table 2, for the micronized insulin ofExample 2 the average particle size D50 was 1.54 μm, the averageparticle size D10 was 0.75 μm, and the average particle size D90 was3.04 μm, which is suitable for pulmonary delivery. D50 is the maximumparticle diameter below which 50 vol % of the sample, based on the totalvolume of the sample, has a smaller particle diameter than the D50particle diameter and above which 50 vol % of the sample has a largerparticle diameter than the D50 particle diameter. D10 is the particlediameter at which 10 vol % of the particles, based on the total volumeof the particles, have a smaller particle diameter than the D10 particlediameter. D90 is the particle diameter at which 90 vol % of theparticles, based on the total volume of the particles, have a smallerparticle diameter than the D90 particle diameter.

TABLE 2 Particle Size Measurement for Example 2 Particle SizeDistribution (μm) Volume Mean Batch No. D10 D50 D90 Diameter Batch 10.72 1.46 2.9 1.68 Batch 2 0.77 1.58 3.0 1.77 Batch 3 0.75 1.52 2.951.74 Batch 4 0.76 1.59 3.31 1.96 Average 0.75 1.54 3.04 1.79 StandardDeviation 0.02 0.06 0.18 0.12 Relative Standard 2.9% 3.9% 6.1% 6.8%Deviation

The chemical stability of the insulin before and after processing wastested by high performance liquid chromatography (HPLC) according toChapter <621> of United States Pharmacopeia (USP) and USP methods usedfor impurity test for the human insulin monograph. FIG. 4 is a chartshowing an impurity profile of insulin before and after the micronizingprocess according to Example 2. As can be seen in FIG. 4, there is not astatistically significant change in the quantity of impurities, such asinsulin dimers, high molecular weight proteins, A-21 desamido insulin orrelated compounds in the insulin during the micronizing process.

FIG. 5 is a high-performance liquid chromatography (HPLC) chromatographof dissolved insulin particles prepared as described in Example 2. TheHPLC chromatograph of FIG. 5 shows that the retention time formicronized insulin does not exhibit a statistically significant changewith respect to that of the original insulin raw material. The evidencefrom the analysis of the micronized insulin particles indicates that thechemical integrity of the insulin is maintained or substantiallymaintained during the micronization process.

The particle size distribution of the micronized insulin particles wasevaluated using a laser diffraction CUVETTE CUV-50ML/US instrument fromSympatec Gmbh. The micronized insulin particles were tested in ethanolmedia (an ethanol solution). The data indicates that the average of thevolume mean diameter for all four (4) batches is 1.79 μm, as shown inTable 2. FIG. 6 is a chart showing Andersen Cascade Impactor studies ofthe human insulin (API produced as described with respect to Example 2)delivered from three metered dose inhalers utilizing a propellantincluding 1,1,1,2-tetrafluoroethane (HFA 134A),1,1,1,2,3,3,3,-heptafluoropropane (HFA 227), or a mixture of HFA 134Aand HFA 227, respectively. The metered dose inhalers were prepared asdescribed below with respect to Example 11. It was concluded from thedata shown in FIG. 6 that the three different propellants (HFA 134A, HFA227, and the mixture of HFA 134A and HFA 227) provided comparableresults when utilized with the micronized human insulin produced asdescribed with respect to Example 2.

FIG. 7 is a chart further showing the Andersen Cascade Impactoranalytical results at three different stage classifications for thehuman insulin (API produced as described with respect to Example 2)delivered from metered dose inhalers utilizing the three differentpropellants (HFA 134a, HFA 227, or a mixture of HFA 134A and HFA 227,respectively). The metered dose inhalers were prepared as describedbelow with respect to Example 11. It was concluded from the data shownin FIG. 7 that the three different propellants provided comparableresults when utilized with the micronized human insulin produced asdescribed with respect to Example 2, for a pulmonary delivery ofinsulin.

Example 3. Method of Preparation of Inhalable Insulin Particles in a 100Vol % Water Solution

Inhalable human insulin particles were prepared as described withrespect to Example 1, except that a roughly 100 vol % purified watersolution having a pH of 2.0 (a solution including purified water and anacid in amount sufficient to provide a pH of 2.0) was used to replacethe acidic solution including 90 vol % of methanol of Example 1. Theparticle size distribution of the resultant inhalable human insulinparticles was analyzed as described with respect to Example 2. Theresults of the particle size distribution analysis showed that theinhalable human insulin particles had a volume mean diameter of 2.01 μm.As noted above, the inhalable human insulin particles prepared asdescribed with respect to Example 1 had a particle size D50 of less than2 μm, and the average of the volume mean diameter of all 4 batches ofthe inhalable human insulin particles prepared as described with respectto Example 2 (which were also prepared using an acidic solutionincluding 90 vol % methanol) was 1.79 μm. Thus, it can be seen that thecomposition of the solvent (e.g. methanol vs. water) can change the sizeof the micronized human insulin that is produced.

Example 4. Methods of Preparation of Inhalable Human Insulin Particlesin Low Methanol Concentration Solution

Inhalable human insulin particles were prepared as described withrespect to Example 1, except that an acidic solution including 50 vol %methanol at a pH of about 2.0 (the other 50 vol % including water andHCl) or an acidic solution including 10 vol % methanol (the other 90 vol% including water and HCl), based on the total volume of the acidicsolution, was used to replace the acidic solution including 90 vol %methanol utilized to dissolve the human insulin raw material of Example1.

Table 3 shows particle size distribution data of human insulin particlesmicronized as described with respect to Examples 1, 3 and 4.

TABLE 3 Particle Size Distributions for examples 1, 3, and 4 ParticleSize Distribution (μm) Volume Mean ID# Solvent D10 D50 D90 DiameterExample 3 100 vol % water 0.65 1.63 3.92 2.01 Example 4 10 vol % MeOH0.65 1.66 3.77 2.0 Example 4 50 vol % MeOH 0.33 0.74 1.52 0.87 Example 190 vol % MeOH 0.72 1.51 2.94 1.71

It was therefore concluded that the starting solvent (e.g., methanolsolution vs. water) and solvent concentration (e.g., methanolconcentration of 10 vol %, 50 vol % or 90 vol %, based on the totalvolume of the acidic solution) utilized to dissolve human insulin (rawmaterial) may affect the particle size of the micronized human insulinparticles.

Example 5. Methods of Preparation of Inhalable Human Insulin Particlesin a 10 Vol % Ethanol Solution

Inhalable human insulin particles were prepared as described withrespect to Example 1, except that an acidic solution including 10 vol %ethanol (the other 90 vol % including water and HCl) having a pH of 2based on the total volume of the acidic solution, was used to replacethe acidic solution including 90 vol % methanol utilized to dissolve theinsulin of Example 1. The particle size distribution of the resultantinhalable human insulin particles was analyzed as described with respectto Example 2. The results of the particle size distribution analysisshowed that the inhalable human insulin particles had a volume meandiameter of 1.36 μm.

Example 6. Method for Micronizing Human Insulin to Inhalable ParticlesUtilizing a 90 Vol % Methanol Solution at a Different pH

Inhalable human insulin particles were prepared as described withrespect to Example 1, except that instead of utilizing a buffer solutionhaving a pH of 5.64 a series of buffer solutions including NaOH having apH of 3 to 9 were utilized. The particle size distributions of theresultant inhalable human insulin particles were analyzed as describedwith respect to Example 2. NaOH was used to adjust the solution pH aswell. The results of the particle size distribution analyses and the pHof the corresponding buffer solution after titration are shown in Table4. It was concluded from the data shown in Table 4 that utilizing abuffer solution having a pH of 3 to 9 is suitable for embodiments of themicronization process. The data obtained shows that over 99 vol % of theparticles, based on the total volume of the particles, have a particlesize smaller than 5 μm. Thus, the micronized insulin particles mayinclude 99 vol % or more (e.g., 99 to 100 vol %) of particles having aparticle size of less than 5 μm, based on the total volume of themicronized insulin particles. In these embodiments, the micronizedinsulin particles may include up to 99 vol % of particles having aparticle size of less than 5 μm, based on the total volume of themicronized insulin particles.

TABLE 4 Insulin Particle Size, Generated at Various pH Particle SizeDistribution (μm) Volume Mean # pH D10 D50 D90 D99 Diameter 1 3.1 0.51.16 2.32 3.69 1.31 2 5.3 0.63 1.29 2.4 3.66 1.42 3 6.0 0.7 1.44 2.274.46 1.63 4 6.2 0.72 1.51 2.94 4.81 1.71 5 7.0 0.6 1.22 2.21 3.51 1.33 67.9 0.56 1.17 2.12 3.41 1.28 7 8.8 0.57 1.18 2.13 3.38 1.29

Example 7. Method of Preparation of Inhalable Human Insulin ParticleUtilizing an Isopropyl Alcohol Co-Solvent

Inhalable human insulin particles were prepared as described withrespect to Example 1, except that isopropyl alcohol was used to replacethe ethanol of Example 1 that was added to the suspension after thetitration was completed or substantially completed. The particle sizedistribution of the resultant inhalable human insulin particles wasanalyzed as described with respect to Example 2. The results of theparticle size distribution analysis showed that the volume mean diameterof the inhalable human insulin particles was 1.27 μm.

Example 8. Method of Preparation of Inhalable Human Insulin ParticleUtilizing an Acetone Co-Solvent

Inhalable human insulin particles were prepared as described withrespect to Example 1, except that acetone was used to replace theethanol of Example 1 that was added to the suspension after thetitration was completed or substantially completed. The particle sizedistribution of the resultant inhalable human insulin particles wasanalyzed as described with respect to Example 2. The results of theparticle size distribution analysis showed that the volume mean diameterof the inhalable human insulin particles was 1.32 μm.

Example 9. Method for Micronizing Insulin Glargine Analogue to InhalableParticles

Insulin glargine is a long acting human insulin analogue. The insulinglargine used here was obtained by ultrafiltration of commerciallyavailable insulin glargine) (LANTUS®. The insulin glargine was washedand lyophilized before use. 70 mg of the washed and lyophilized insulinglargine was dissolved in 7.7 ml of an acidic solution having a pH ofabout 2.2, a mixture of 90 vol % methanol and 10 vol % water, based onthe total volume of the acidic solution, to form a dissolved insulinsolution including an insulin glargine. 1.75 ml of a phosphate buffersolution having a pH of 6.9 was added dropwise to titrate the dissolvedinsulin glargine solution after the insulin glargine was completelydissolved. 10 ml of ethanol was added to the solution. The foregoingdissolving, titrating, and addition of ethanol were performed understeady (substantially continuous) stirring. The clear dissolved insulinglargine solution becomes a milky suspension including micronizedinsulin glargine particles (micronized insulin glargine particles). Themicronized insulin glargine particles were separated, washed and dried.The particle size distribution of the micronized insulin glargineparticles was analyzed using the laser diffraction test described withrespect to Example 2. The particle distribution analysis showed that thevolume mean diameter of the micronized insulin glargine particles was2.27 μm. FIG. 8 is a Scanning Electron Microscopy (SEM) image of themicronized insulin glargine particles. FIG. 9 is an HPLC chromatographof the dissolved micronized insulin glargine particles. Retention timeof the HPLC results shown in FIG. 9 indicates that the chemicalproperties of the insulin glargine did not change (or did notsubstantially change) during the micronization process. FIGS. 10 and 11are charts showing the results of an Andersen Cascade Impactor study ofthe insulin glargine particles delivered from metered dose inhalersutilizing HFA 134A as a propellant. The metered dose inhalers wereprepared as described below with respect to Example 11. The studyresults shown in FIGS. 10 and 11 demonstrated a consistent orsubstantially consistent pattern for a pulmonary delivery of insulin.

Example 10. Method for Micronizing Insulin Aspart Analogue to InhalableParticles

Insulin Aspart is a fast-acting insulin analogue. Insulin Aspart usedhere was obtained by ultrafiltration of NovoLog® (obtained from NovoNordisk, Bagsværd, Denmark). The ultrafiltered insulin aspart was washedand lyophilized before use. 70 mg of washed and lyophilized insulinaspart was dissolved in 7.7 ml of an acidic water solution having a pHof about 2 and including HCl to form a dissolved insulin solutionincluding insulin aspart. 4.2 ml of an acetate buffer solution having apH of 5.64 was added dropwise to titrate the dissolved insulin aspartsolution after the insulin aspart was completely dissolved. 78 ml ofethanol was added to the solution to obtain a suspension. The foregoingdissolving, titrating, and addition of ethanol were performed understeady (substantially continuous) stirring to stabilize the particlesbefore wash. The clear dissolved insulin aspart solution became a milkysuspension including micronized insulin aspart particles (micronizedinsulin aspart particles). The micronized insulin aspart particles wereseparated, washed and dried. The particle size distribution of themicronized insulin aspart particles was analyzed using the laserdiffraction test described with respect to Example 2. The particledistribution analysis showed that the volume mean diameter of themicronized insulin aspart particles was 2.72 μm. FIG. 12 is a ScanningElectron Microscopy (SEM) image of the micronized insulin aspartparticles. FIG. 13 is an HPLC chromatograph of the dissolved micronizedinsulin aspart particles. Retention time of the HPLC results shown inFIG. 13 indicates that the chemical properties of the insulin aspart didnot change (or did not substantially change) during the micronizationprocess.

FIGS. 14 and 15 are charts showing the results of an Andersen CascadeImpactor study of the insulin aspart particles delivered from metereddose inhalers utilizing HFA 134A as a propellant. The metered doseinhalers were prepared as described below with respect to Example 11.The study results shown in FIGS. 14 and 15 demonstrated a consistent orsubstantially consistent pattern for a pulmonary delivery of insulin.

Example 11. Preparation of Metered Dose Inhalers for In Vitro AndersenCascade Impactor Tests

Metered dose inhalers (MDIs) were prepared according to the followingprocess. A suitable or appropriate amount of micronized human insulinAPI (e.g., micronized human insulin particles or micronized humaninsulin analogue particles) and ethanol were filled into an inhalercanister. The contents of the canister were then mixed by applyingultrasonic energy using a VWR Aquasonic for 5 minutes to achieve auniform or substantially uniform suspension. Different propellants suchas HFA 134A, HFA 227 or a mixture thereof were added, and the canisterwas sealed utilizing a suitable valve by clamping.

Micronized human insulin (e.g., micronized human insulin particles ormicronized insulin analogue particles) was filled into the metered doseinhaler (MDI) as the active ingredient. The concentration of humaninsulin or insulin analogue in the inhaler was 3 mg/g. The AndersenCascade Impactor data shown in FIG. 7, FIG. 11, and FIG. 15 correspondwell with the particle size distribution results observed utilizing alaser diffraction particle size analyzer. In the Andersen CascadeImpactor data provided herein, emitted dose refers to the percentage ofthe human insulin or insulin analogue that was deposited on the AndersenCascade Impactor.

The shape and roughness (or smoothness) of the surface of the humaninsulin particles micronized by embodiments of the process disclosedherein is quite suitable or favorable (e.g., suitable or favorable forpulmonary delivery). Micronization by jet milling is a common way togrind particles from a millimeter size range to a smaller micrometersize range. The jet milling process involves frequent collisions amongthe particles as well as collisions with a wall of a milling chambercaused by a high speed gas stream. The micronized particles produced byjet milling are extracted from the milling chamber by a circular motionof a gas stream and centrifugal forces. These mechanical forces maydamage the surface and the shape of the micronized particles, forexample, as described below with respect to Comparative Example 1, whichmay not be favorable or suitable for pulmonary delivery.

Example 12. High Purity Micronized Inhalable Insulin Particles Suitablefor Pharmaceutical Application

One (1) gram of biosynthetic human insulin API powder (i.e., recombinantinsulin from Amphastar France Pharmaceuticals S.A.S.) was dissolved in110 ml of an acidic solution having a pH of about 1.9 in a mixture of90% by volume of methanol and 10% by volume of water in a 400 ml beakerwith a centrifugal stirrer or stirring bar. The resultant solution wasstirred until the insulin solution was completely dissolved orsubstantially completely clear to form insulin true solution. Then, thestirring was slowed to a slower mode (e.g., a spinning speed of about 50rpm), and 25 ml of a 0.1 M NaAc/HAc buffer solution (having a pH of5.64) was added dropwise to titrate the dissolved insulin solution. Theclear dissolved insulin solution turned into a milky and yellowishsuspension including micronized insulin particles. After the titrationwas completed or substantially completed, about 135 ml of ethanol (no pHadjusted) was added to the suspension, and the stirring was continuedfor another 30 minutes for stabilizing the particle and higher yield.

The micronized insulin particles were separated from the supernatant ofthe suspension by ultrafiltration and the solid was washed with ethanoltwice to remove methanol and salt. The wash and concentration processmay be repeated if the solvent impurities are high. The solid was vacuumdried at room temperature. The washing process and concentration ofinsulin were carried out as described with respect to Example 2 above.The particle size for the resultant micronized insulin is 1.61 μm VMD.

More lots were made with 0.5 g batch for purity and impurity tests usingthe same process.

The purity and impurity profile of the obtained three (3) batches ofinsulin particles are tested and listed in Table 5 as follows at drybase (e.g., the results are shown as % by weight relative to the totalweight of the insulin particles on a dried basis).

TABLE 5 Test Results Purity and Impurity Profile of Insulin ParticlesInsulin-related inpurities Solvent- Non-Solvent A-21 Related High MWimpurity impurity Batch # Purity Desamido Substance proteins TotalMethanol Acetate 1 98.7% 0.39% 1.1% 0.32% 1.8% <0.001% 0.07% 2 98.8%0.36% 1.1% 0.30% 1.7% <0.001% 0.04% 3 98.8% 0.34% 1.1% 0.30% 1.7% 0.009%0.03% Average 98.8% 0.36% 1.1% 0.31% 1.8% 0.009% 0.05%

The data in Table 5 shows all three (3) lots:

-   -   The purity of the obtained Insulin particles is above 98% (e.g.,        the purity of insulin in the insulin particles is above 98% by        weight, based on the total weight of the insulin particles on a        dried basis);    -   A21 desamido content in the final product was less than 0.40%        (e.g., 0.40% by weight based on the total weight of the insulin        particles on a dried basis), and micronized insulin's related        compounds was around 1.1% (e.g., 1.1% by weight based on the        total weight of the insulin particles on a dried basis), less        than the 2% by weight of USP criteria;    -   High molecular weight proteins in human insulin was around 0.3%        (e.g., 0.3% by weight based on the total weight of the insulin        particles on a dried basis), less than USP criteria of 1%;    -   The amount of methanol solvent impurity was no more than 0.009%        (e.g., 0.009% by weight based on the total weight of the insulin        particles on a dried basis);    -   The amount of acetate non-solvent impurity (acetate salt) was no        more than 0.07% (e.g., 0.07% by weight based on the total weight        of the insulin particles on a dried basis); and    -   The purity of the resulting insulin particles is more than 98%        (e.g., the purity of insulin in the insulin particles is above        98% by weight, based on the total weight of the insulin        particles on a dried basis).

Example 13. Controlled Zinc Content in Micronized Inhalable InsulinParticles

Seventy (70) mg of biosynthetic human insulin (i.e. recombinant insulinavailable from Amphastar France Pharmaceuticals S.A.S.) raw materialpowder was dissolved in 7.7 ml of an acidic solution having a pH ofabout 1.9 a mixture of 90% by volume of methanol and 10% by volume ofwater in a 40 ml vial. The vial was placed on top of a stirring plateand the resultant solution was steadily stirred until the solution wascompletely dissolved or substantially clear to form insulin truesolution. Then, the stirring was slowed to a slower mode (e.g., aspinning speed of about 75 rpm), and 1.75 ml of a 0.1 M sodiumacetate/acetic acid (NaAc/HAc) buffer solution having a pH of 5.64 wasadded dropwise to slowly titrate the dissolved insulin solution. Theclear dissolved insulin solution turned into a milky and yellowishsuspension including micronized insulin particles. About 10 ml ofethanol was added to the suspension after the titration was completed orsubstantially completed. The stirring was continued for another 30minutes for stabilizing the particles and to provide higher yield. Themicronized insulin particles were separated from a supernatant of thesuspension as a solid by centrifuge. The solid was washed with ethanoltwice to remove methanol and salt. The solid was vacuum dried at roomtemperature.

The zinc content in the micronized insulin has no significant changesduring the process:

-   -   The zinc content before the process in the insulin is 0.39%        (e.g., 0.39% by weight based on the total weight of the        biosynthetic human insulin),    -   The zinc content after the process in the micronized insulin is        0.42% (e.g., 0.42% by weight based on the total weight of the        insulin particles on a dried basis).

Accordingly, in embodiments of the disclosure, the zinc content will bebelow 1% (e.g., below 1% by weight based on the total weight of theinsulin particles on a dried basis) and higher than 0.3% (e.g., higherthan 1% by weight based on the total weight of the insulin particles ona dried basis) in the micronized particle if Human Insulin USP was usedas starting material.

Example 14. Insulin Micronization Process with Higher Recovery Rate

One (1) gram of biosynthetic human insulin API powder (i.e., recombinantinsulin from Amphastar France Pharmaceuticals S.A.S.) was dissolved in110 ml of an acidic solution having a pH of about 1.9 in a mixture of90% by volume of methanol and 10% by volume of water in a 400 ml beakerwith a centrifugal stirrer or stirring bar. The resultant solution wasstirred until the insulin solution was completely dissolved orsubstantially completely clear to form insulin true solution. Then, thestirring was slowed to a slower mode (e.g., a spinning speed of about 50rpm), and 25 ml of a 0.1 M NaAc/HAc buffer solution (having a pH of5.64) was added dropwise to titrate the dissolved insulin solution. Theclear dissolved insulin solution turned into a milky and yellowishsuspension including micronized insulin particles. After the titrationwas completed about 135 ml of ethanol at 2-8° C. was added to thesuspension. The stirring was continued for another 30 minutes under 2-8°C. for stabilizing the particle size and higher yield.

The micronized insulin particles were separated from the supernatant ofthe suspension as a solid by ultrafiltration and the solid was washedwith ethanol twice to remove methanol. The obtained solid of micronizedinsulin was vacuum dried at room temperature. A mass of 0.92 gram ofmicronized insulin was obtained. The recovery rate of Insulin with thisprocess is 92% (e.g., the yield of the insulin particles was 92% byweight based on the total weight of the final product).

Example 15. Insulin Micronization Process Compatible with MDIFormulation and Compounding

One (1) gram of biosynthetic human insulin API powder (i.e., recombinantinsulin from Amphastar France Pharmaceuticals S.A.S.) was dissolved in110 ml of an acidic solution having a pH of about 1.9 in a mixture of90% by volume of methanol and 10% by volume of water in a 400 ml beakerwith a centrifugal stirrer or stirring bar. The resultant solution wasstirred until the insulin solution was completely dissolved orsubstantially completely clear to form insulin true solution. Then, thestirring was slowed to a slower mode (e.g., a spinning speed of about 50rpm), and 25 ml of a 0.1 M NaAc/HAc buffer solution (having a pH of5.64) was added dropwise to titrate the dissolved insulin solution. Theclear dissolved insulin solution turned into a milky and yellowishsuspension including micronized insulin particles. After the titrationwas completed, about 135 ml of ethanol at 2-8° C. was added to thesuspension. The stirring was continued for another 30 minutes at atemperature of 2-8° C. for stabilizing the particle size and resultingin a higher yield before wash.

The micronized insulin particles were concentrated from the supernatantof the suspension by ultrafiltration with 500 kD modifiedpolyethersulfone membrane from Spectrum Lab at Rancho Dominguez, Calif.The suspension of insulin particles was concentrated to 5-10 ml, whichwas washed with 100 ml ethanol three times to remove impurities, likemethanol and salt.

The metered dose inhaler, or MDI product (a pulmonary product) isreadily processed and compounded with the obtained insulin particles.

Comparative Example 1. Preparation of Human Insulin Particles Via JetMilling

Human Insulin particles were prepared by jet milling utilizing agrinding N₂ pressure of 75 PSI and a feeding rate about 1 g/min. FIG. 16is an atomic force microscopy (AFM) image of human insulin particlesthat were micronized using the jet milling method. As can be seen in theimage of FIG. 16, the human insulin particles prepared by jet millinghave a rough and irregular (or uneven) appearance.

FIG. 17 is an AFM image of inhalable human insulin particles micronizedas described with respect to Example 2. Since embodiments of the processdisclosed herein are carried out at room temperature and involve nomechanical forces and/or heat (or substantially no mechanical forcesand/or heat), the micronized human insulin particles have a shape andsurface that are more suitable or more preferred for human pulmonarydelivery.

According to an embodiment, a high-purity inhalable insulin material fora pulmonary pharmaceutical product, includes: insulin particles having aparticle size at the micrometer level, and having the followingcharacteristics: (i) the purity of the insulin particles being not lessthan 96% by weight on the dried basis (e.g., insulin being present inthe insulin particles in an amount of not less than 96% by weight basedon the total weight of the insulin particles on a dried basis); (ii)optionally, the amount of zinc in the insulin particles being not morethan 1%, and not less than 0.3% by weight, on a dried basis (e.g., notmore than 1% by weight and not less than 0.3% by weight based on thetotal weight of the insulin particles on a dried basis); (iii) the totalamount of insulin-related impurities in the insulin particles being notmore than 2% by weight (e.g., not more than 2% by weight based on thetotal weight of the insulin particles on a dried basis); (iv) the totalamount of solvent-related impurities in the insulin particles, whichsolvent-related impurities do not include a co-solvent formulationcomponent in the pulmonary product, being not more than 0.03% by weight(e.g., not more than 0.03% by weight based on the total weight of theinsulin particles on a dried basis); and the total amount ofnonsolvent-related impurities in the insulin particles being not morethan 0.3% by weight (e.g., not more than 0.3% by weight based on thetotal weight of the insulin particles on a dried basis). The insulinparticles may further include any or all of the followingcharacteristics.

The micronized insulin particles may include an insulin selected fromhuman insulin, an animal insulin, an insulin analogue, and any mixturethereof.

The insulin analogue may be selected from insulin aspart, insulinglargine, and any mixture thereof.

The insulin particles may be substantially spherical (e.g., generallyspherical) in shape and have a particle size of less than 5 μm.

99% by volume of the insulin particles may include particles having aparticle size of less than 5 μm, based on the total volume of themicronized insulin particles.

The insulin particles may include substantially spherical (e.g.,generally spherical) particles having a volume mean particle diameter(e.g., D50) of about 1 μm to 2 μm.

According to an embodiment, a high-efficiency method of preparing ahigh-purity inhalable insulin having a particle size at the micrometerlevel suitable for a pharmaceutical pulmonary product, the methodincluding: (1) dissolving an insulin raw material in an acidic solutionto form a dissolved insulin solution; (2) titrating the dissolvedinsulin solution with a buffer solution to form a suspension includinginsulin particles (e.g., micronized insulin particles) having a particlesize at the micrometer level; (3) stabilizing the insulin particles(e.g., the micronized insulin particles) with a solvent; (4)concentrating the suspension including the insulin particles (e.g., themicronized insulin particles); and washing the suspension with a solventand further concentrating the suspension, where the resultant washed andconcentrated suspension including the micronized insulin particles isready and suitable for further processing to form a pulmonary product,and where the method results in (e.g., provides) the insulin particleshaving the particle size at the micrometer level, in which 99% (e.g.,99% by volume) of the insulin particles (e.g., the micronized insulinparticles) have a particle size less than 5 μm and the volume meanparticle diameter (e.g., D50) of the insulin particles (e.g., themicronized insulin particles) is about 1 μm to 2 μm, and in which thepurity of the insulin particles is not less than 96% on the dried basis(e.g., an amount of insulin in the insulin particles is not less than96% by weight based on the total weight of the insulin particles on adried basis); optionally, the amount of zinc in the insulin particles isnot more than 1% (e.g., not more than 1% by weight based on the totalweight insulin particles on a dried basis), and not less than 0.3% byweight on the dried basis (e.g., based on the total weight of theinsulin particles on a dried basis); the total amount of allinsulin-related impurities in the insulin particles is not more than 2%(e.g., not more than 2% by weight based on the total weight of theinsulin particles on a dried basis); the total amount of solvent-relatedimpurities in the insulin particles, which solvent-related impurities donot include a co-solvent formulation component in the pulmonary product,is not more than 0.03% (e.g., not more than 0.03% by weight based on thetotal weight of the insulin particles on a dried basis); and the totalamount of nonsolvent-related impurities in the insulin particles is notmore than 0.3% (e.g., not more than 0.3% by weight based on the totalweight of the insulin particles on a dried basis). The method mayfurther include any or all of the following.

The insulin raw material may include a crystalline insulin selected fromcrystalline human insulin, crystalline animal insulin, crystallineinsulin analogue (e.g., a crystalline human insulin analogue), and anymixture thereof.

The crystalline insulin analogue may be selected from the groupconsisting of crystalline insulin aspart, crystalline insulin glargine,and any mixture thereof.

The acidic solution may include water and methanol.

The acidic solution may include methanol in an amount of 10% to 90% byvolume, based on the total volume of the acidic solution.

The acidic solution may have a pH in the range of 1 to 3.

The acidic solution may have a pH in the range of 1.5 to 2.5.

The acidic solution may be prepared at room temperature.

The titrating of the acidic solution is performed using a buffersolution including sodium acetate and acetic acid.

The titrating may be performed at a pH in the range of 3 to 9.

The titrating may be performed at a pH in the range of 4.5 to 7.5.

The titrating may be performed at room temperature.

The stabilizing may include adding a stabilizing agent including ethanolto the suspension.

The volume of the stabilizing agent including ethanol may be 0.5 to 2times larger than the volume of the insulin solution (e.g., thedissolved insulin solution).

The stabilizing agent including ethanol may have a neutral pH.

The stabilizing agent including ethanol may be at temperature in therange of 0 to 25° C.

In some embodiments, the adding of the stabilizing agent increases theyield of the insulin particles.

The washing of the suspension including the insulin particles mayinclude washing the suspension with ethanol.

The ethanol may be at a temperature of 0 to 25° C.

The suspension may be a concentrated suspension.

In some embodiments, the resultant concentrated suspension including theinsulin particles and the ethanol, and dried insulin particles preparedfrom the resultant concentrated suspension, are ready for the furtherprocessing to form the pulmonary product.

The washing and concentrating may be repeated multiple times (e.g., aplurality of times).

The yield of the inhalable insulin particles may be 75% or greater(e.g., the yield of the inhalable insulin particles may be 75% by weightor greater, based on the total amount of the final product, for example,on a dried basis).

The yield of the inhalable insulin particles may be 85% by weight orgreater (e.g., the yield of the inhalable insulin particles may be 85%by weight or greater, based on the total weight of the final product).

While the present invention has been described in connection withcertain embodiments, it is to be understood that the invention is notlimited to the disclosed embodiments, but, on the contrary, is intendedto cover various modifications and equivalent arrangements includedwithin the spirit and scope of the appended claims, and equivalentsthereof. Throughout the text and claims, the terms “about” and“substantially” are used as terms of approximation, not terms of degree,and reflect the inherent variation associated with measurement,significant figures, and interchangeability, all as understood by aperson having ordinary skill in the relevant art. Also, it is to beunderstood that throughout this disclosure and the accompanying claims,even values that are not preceded by the term “about” are alsoimplicitly modified by that term, unless otherwise specified.

What is claimed is:
 1. A high-purity inhalable insulin material for apulmonary pharmaceutical product to be delivered by metered doseinhaler, comprising: insulin particles having a particle size at themicrometer level, and having the following characteristics: (i) thepurity of the insulin particles being not less than 96% by weight on thedried basis; (ii) the amount of zinc in the insulin particles being notmore than 1% by weight, and not less than 0.3% by weight, on the driedbasis; (iii) the total amount of insulin-related impurities consistingof insulin dimer, high molecular weight proteins, and A-21 desamidoinsulin in the insulin particles being not more than 2% by weight on thedried basis, wherein, of the total amount of insulin-related impurities,the total amount of A-21 desamido insulin is less than or equal to0.63%, and/or the total amount of insulin dimer is less than or equal to0.13%, by weight on the dried basis; (iv) the total amount ofsolvent-related impurities in the insulin particles, whichsolvent-related impurities do not include a co-solvent formulationcomponent in the pulmonary product, being not more than 0.03% by weighton the dried basis; and (v) the total amount of nonsolvent-relatedimpurities other than insulin-related impurities in the insulinparticles being not more than 0.3% by weight on the dried basis.
 2. Theinsulin material of claim 1, wherein the micronized insulin particlescomprise an insulin selected from the group consisting of human insulin,an animal insulin, an insulin analogue, and any mixture thereof.
 3. Theinsulin material of claim 2, wherein the insulin analogue is selectedfrom the group consisting of insulin aspart, insulin glargine, and anymixture thereof.
 4. The insulin material of claim 1, wherein the insulinparticles are substantially spherical in shape and have a particle sizeof less than 5 μm.
 5. The insulin material of claim 1, wherein 99% byvolume of the insulin particles comprise particles having a particlesize of less than 5 μm, based on the total volume of the micronizedinsulin particles.
 6. The insulin material of claim 1, wherein theinsulin particles comprise substantially spherical particles having avolume mean particle diameter of about 1 μm to 2 μm.
 7. A high-purityinhalable insulin material for a pulmonary pharmaceutical product to bedelivered by metered dose inhaler, consisting of: unencapsulated insulinparticles having a particle size at the micrometer level, and having thefollowing characteristics: (i) the purity of the unencapsulated insulinparticles being not less than 96% by weight on the dried basis; (ii) theamount of zinc in the unencapsulated insulin particles being not morethan 1% by weight, and not less than 0.3% by weight, on the dried basis;(iii) the total amount of insulin-related impurities consisting ofinsulin dimer, high molecular weight proteins, and A-21 desamido insulinin the unencapsulated insulin particles being not more than 2% by weighton the dried basis, wherein, of the total amount of insulin-relatedimpurities, the total amount of A-21 desamido insulin is less than orequal to 0.63%, and/or the total amount of insulin dimer is less than orequal to 0.13%, by weight on the dried basis; (iv) the total amount ofsolvent-related impurities in the unencapsulated insulin particles,which solvent-related impurities do not include a co-solvent formulationcomponent in the pulmonary product, being not more than 0.03% by weighton the dried basis; and (v) the total amount of nonsolvent-relatedimpurities other than insulin-related impurities in the unencapsulatedinsulin particles being not more than 0.3% by weight on the dried basis.8. A metered dose inhaler comprising the high-purity inhalable insulinmaterial of claim 7.